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Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders characterized by dysplastic hematopoiesis in the bone marrow and peripheral blood cytopenias. The splicing factor SF3B1 is the most commonly mutated gene in MDS and is mainly found in refractory anemia with ring sideroblasts (RARS). SF3B1 mutations are missense, heterozygous and clustered, suggesting they are gain-of-function with a causal link to the development of RARS. Nevertheless, in vitro studies are hampered by the inability to clone the full length SF3B1 cDNA and model the effect of its mutation in cell lines. Few reports on Sf3b1 knock-down mice have not convincingly recapitulated the features of its somatic mutations in RARS. 

We therefore targeted the Sf3b1 locus of mouse embryonic stem cells (ESC) to create a conditional allele encoding the most common mutation Sf3b1^K700E to assess the phenotypic consequences of its expression in vitro and in vivo.

WT and mutant Sf3b1 ESC showed proliferation rates and colony morphologies similar to native ESC. Upon differentiation, >80% of cells expressed the pan-hematopoietic marker Cd45, with no difference between genotypes.  Erythroid differentiation in particular was efficient as confirmed by an increase in transcription levels of the globin genes, that was reduced in Sf3b1 mutant cells. Nevertheless, gross morphology was similar in WT and mutant cells. Flow cytometry showed a marginal decrease in the percentage of Cd71^low/Ter119^hi cells for the mutant genotype, but no major differences in the frequency of expression markers of stem and progenitor, myeloid and megakaryocytic cells.

By unsupervised clustering of gene expression profiles, we found that WT and mutant cells cluster closely together in the undifferentiated state, whereas differentiation causes mutant cells to acquire a dramatically different expression profile than WT cells. Mutant differentiated cells showed an excess of downregulated genes, among which known players in congenital sideroblastic anemias (Abcb7, Glrx5, and Scl25A38). Gene ontology analysis showed downregulation of several biological processes, including the respiratory electron transport chain and regulators of mitochondrial ion transport as shown previously, but also processes involved in RNA metabolism like translation initiation and pre-mRNA splicing.

Initial analysis of conditional Sf3b1^K700E expression in mice showed that heterozygous animals display no overt sign of disease at our median follow-up of 116 days. In turn, peripheral blood counts post-pIpC injection show that hemoglobin is significantly decreased at one (17.2 vs 14.2 gr/dl, p=0.001) and two months (16.7 vs 15, p=0.006) in mutant animals, with no difference in white cell and platelet counts. Analysis of bone marrow cells by flow cytometry showed a 2-fold decrease in Cd71^low/Ter119^hi (p=0.02) cells for the mutant genotype, suggestive of a late maturation defect. Lastly, iron stain of bone marrow cells showed an increment of iron-laden macrophages and occasional ringed sideroblasts in the mutant animals.

Our data closely recapitulates initial observations in human samples and suggests mutated Sf3b1 expression leads to defective erythroid maturation. Our mouse model will allow better characterization of the molecular pathways altered by the mutation and the analysis of modifiers of this phenotype.